Desiccation and Zinc Binding Induce Transition of Tomato ... · drought and salinity (Yang et al., 2005). Maize (Zea mays ) ASR1 was proposed as a candidate gene for quantitative

Desiccation and Zinc Binding Induce Transition ofTomato Abscisic Acid Stress Ripening 1, a WaterStress- and Salt Stress-Regulated Plant-Specific Protein,from Unfolded to Folded State1[W][OA]

Yehuda Goldgur2, Slava Rom, Rodolfo Ghirlando, Doron Shkolnik, Natalia Shadrin, Zvia Konrad,and Dudy Bar-Zvi*

Department of Chemistry (N.S, Y.G.) and Department of Life Sciences and Doris and Bertie Black Center forBioenergetics in Life Sciences (D.B.-Z., D.S., S.R., Z.K.), Ben-Gurion University, Beer-Sheva 84105, Israel; andLaboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, NationalInstitutes of Health, Department of Health and Human Services, Bethesda, Maryland 20892–0540 (R.G.)

Abscisic acid stress ripening 1 (ASR1) is a low molecular weight plant-specific protein encoded by an abiotic stress-regulatedgene. Overexpression of ASR1 in transgenic plants increases their salt tolerance. The ASR1 protein possesses a zinc-dependentDNA-binding activity. The DNA-binding site was mapped to the central part of the polypeptide using truncated forms of theprotein. Two additional zinc-binding sites were shown to be localized at the amino terminus of the polypeptide. ASR1 proteinis presumed to be an intrinsically unstructured protein using a number of prediction algorithms. The degree of order of ASR1was determined experimentally using nontagged recombinant protein expressed in Escherichia coli and purified to homogeneity.Purified ASR1 was shown to be unfolded using dynamic light scattering, gel filtration, microcalorimetry, circular dichroism, andFourier transform infrared spectrometry. The protein was shown to be monomeric by analytical ultracentrifugation. Addition ofzinc ions resulted in a global change in ASR1 structure from monomer to homodimer. Upon binding of zinc ions, the proteinbecomes ordered as shown by Fourier transform infrared spectrometry and microcalorimetry, concomitant with dimerization.Tomato (Solanum lycopersicum) leaf soluble ASR1 is unstructured in the absence of added zinc and gains structure upon binding ofthe metal ion. The effect of zinc binding on ASR1 folding and dimerization is discussed.

Tomato (Solanum lycopersicum) abscisic acid (ABA)stress ripening 1 (ASR1) is a highly charged, low Mrprotein whose expression is regulated by salt andwater stress and by the plant hormone ABA (Iusemet al., 1993; Amitai-Zeigerson et al., 1995). ASR1 be-longs to a plant-specific protein family (Carrari et al.,2004). ASR homologs were cloned from a large num-ber of dicot and monocot plants (summarized inCarrari et al., 2004) and from gymnosperm specieslike pine (Pinus spp.; Padmanabhan et al., 1997) andGinkgo biloba (Shen et al., 2005), and in all cases thehomologs were cloned from vegetative (leaf, shoot,

and stem) and reproductive (fruit and pollen) tissues.Expression levels of a number of ASR genes arerapidly increased in response to salt and drought(Amitai-Zeigerson et al., 1995; Silhavi et al., 1995;Padmanabhan et al., 1997; Riccardi et al., 1998;Kawasaki et al., 2001; Sugiharto et al., 2002; Wanget al., 2003a). Whereas most ASR orthologs are regu-lated by ABA, the expression of the potato (Solanumtuberosum) drought-inducible ortholog DS2 gene isABA independent (Doczi et al., 2005). A number ofASR genes were shown to be developmentally regu-lated and expressed during pollen (Wang et al., 1998),flower (Gilad et al., 1997; Doczi et al., 2005), and fruitdevelopment (Canel et al., 1995; Gilad et al., 1997;Mbeguie-A-Mbeguie et al., 1997; Hong et al., 2002;Cakir et al., 2003; Doczi et al., 2005).

Tomato ASR1 is localized in both cytosol and nucleuscompartments (Kalifa et al., 2004a). Fusion proteins ofASR homologs with reporter proteins were shown to belocalized in nuclei (Cakir et al., 2003; Wang et al., 2003b,2003c). Tomato ASR1 (Kalifa et al., 2004a) and the grape(Vitis vinifera) ortholog VvMSA (maturation-, stress-,and ABA-induced protein; Cakir et al., 2003) were shownto posses DNA-binding activity. The zinc-dependentDNA-binding activity of tomato ASR1 can be obtainedin a carboxy terminus fragment of ASR1 (Rom et al.,2006). Two zinc-binding sites were mapped at theamino terminus of the protein (Rom et al., 2006).

1 This work was supported by the Israel Science Foundation (toD.B.Z. and Y.G.) and in part by the Intramural Research Program ofthe National Institutes of Health, National Institute of Diabetes andDigestive and Kidney Diseases (to R.G.).

2 Present address: X-Ray Crystallography Core Facility, MemorialSloan-Kettering Cancer Center, 1275 York Avenue, New York 10021.

* Corresponding author; e-mail [email protected]; fax 972–8–6479198.

The author responsible for distribution of materials integral to thefindings presented in this article in accordance with the policydescribed in the Instructions for Authors (www.plantphysiol.org) is:Dudy Bar-Zvi ([email protected]).

[W] The online version of this article contains Web-only data.[OA] Open Access articles can be viewed online without a sub-

scription.www.plantphysiol.org/cgi/doi/10.1104/pp.106.092965

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Overexpression of tomato ASR1 protein in tobacco(Nicotiana tabacum) plants results in an increased salttolerance and in the modulation of expression of othergenes (Kalifa et al., 2004b). Overexpression in Arabidop-sis (Arabidopsis thaliana) of the ortholog LLA23 gene fromlily (Lilium longiflorum) increases the plant tolerance todrought and salinity (Yang et al., 2005). Maize (Zea mays)ASR1 was proposed as a candidate gene for quantitativetrait locus for drought stress response (de Vienne et al.,1999). Moreover, the ASR orthologs VvMSA (Cakir et al.,2003) and LLA23 (Yang et al., 2005) were suggested to beinvolved in the expression of sugar-metabolizing genesand in ABA-signaling pathways, respectively.

Although it is a DNA-binding protein, ASR1 does notshare sequence or structure homologies with otherknown DNA-binding proteins (see Kalifa et al., 2004a;Rom et al., 2006). ASR1, a low Mr, highly charged protein,is predicted to be intrinsically unstructured. Recently,intrinsically unstructured/disordered or natively un-folded proteins have become a focus of scientific interest(Uversky, 2002, 2006; Tompa, 2002; Fink, 2005). It isestimated that as many as 30% of eukaryotic proteins areeither completely or partially disordered (Fink, 2005).Folding of intrinsically unstructured proteins is sug-gested to regulate the activity of these proteins. Foldingcan be induced by the binding of cofactor or by protein-protein interaction (Dyson and Wright, 2002). Certainstress proteins have been shown to be intrinsicallyunfolded. For example, some dehydrin proteins thatare expressed at high levels under abiotic stress condi-tions were shown to be unstructured (Eom et al., 1996;Lisse et al., 1996; Soulages et al., 2003; Mouillon et al.,2006). Moreover, dehydrin homolog from a desiccation-tolerant nematode was also shown to be unfolded underphysiological condition (Goyal et al., 2003).

In this study, we demonstrate both by biochemicaland biophysical methods that recombinant, full-size,nontagged ASR1 is disordered under physiologicalconditions. The protein transitions to an ordered stateupon the binding of zinc ions, as demonstrated spec-troscopically and calorimetrically. Fourier transforminfrared (FTIR) studies indicate that folding can alsobe induced by desiccation of the ASR1 preparation. Theoligomeric state of ASR1 was studied by analyticalcentrifugation. ASR1 was found to be monomeric in theabsence of zinc and dimeric in the presence of the metalion. Chemical cross-linking confirmed the ability ofASR1 to form homodimers. Tomato leaf cytosolic ASR1is highly sensitive to protease degradation indicative ofits unstructured nature. Zinc binding results in a de-creased susceptibility of leaf soluble ASR1 to proteaseactivity in agreement with the higher degree of struc-ture induced by its binding to the protein.

RESULTS

Zinc Binding Induces Dimerization of ASR1

Sedimentation equilibrium experiments were car-ried out to determine the oligomeric state of ASR1

protein in solution. At all rotor speeds, the sedimen-tation equilibrium profiles were consistent with thepresence of at least two species having very distinctmolecular masses. Analyses in terms of two idealsolutes lead to excellent data fits (Fig. 1A) with buoy-ant molecular masses [Mi (1 2 vir)] of 3,100 6 210 and41,500 6 2,200 D for the low and high molecular massspecies, respectively. The predominant species is thesmaller mass species whose buoyant molecular masscorresponds to an experimental mass of 11,900 6 840D, indicating that in the absence of added Zn21, ASR1(calculated mass, 13,129.7 D) is monomer in solution.The large aggregate, present as an impurity, had amolecular mass approximately 12 times that expectedfor the monomer. Similar observations were made inthe presence of added 1 mM ZnCl2, except that [M1(1 2 v1r)] 5 6,030 6 200 D was measured. This valuecorresponds to a calculated mass of 23,100 6 770 D,implying that in the presence of Zn21, ASR1 formsdimers (n 5 1.8 6 0.06; Fig. 1B). In addition, loweramounts of a larger species having a molecular massof 60,600 6 2,100 D, consistent with the presence ofsmaller ASR1 aggregates (n 5 4.6 6 0.6), were ob-served in presence of added Zn21. A similar analysison ASR1D61 to 115 showed that in the absence ofadded Zn21, the truncated protein had a calculatedmass of 7,600 6 260 D, indicating that it is monomericin solution (calculated mass, 7,044.8 D). Addition of1 mM ZnCl2 to the protein solution led to the formationof a polydisperse system. The major ASR1 forms weremonomers and dimers, although higher species werealso noted (data not shown).

Chemical cross-linking experiments using ethylene-glycol-bis-[succinimidyl succinate] (EGS) confirmedthat ASR1 and ASR1D61 to 115 form dimers in solution(Fig. 2). No ASR1 and ASR1D61 to 115 cross-linking wasobserved with dimethyl suberimidate and 1,5-difluoro-2,4-dinitrobenzene (data not shown). These cross-linkingreagents used react with amino groups and differ inthe length of the spacer arm (16.1 A, 11.0 A, and 3.0 Afor EGS, dimethyl suberimidate, and 1,5-difluoro-2,4-dinitrobenzene, respectively). The observed cross-linker specificity suggests that the cross-linked specieshad an ordered structure and were unlikely to repre-sent aggregated or inflexible structures.

Apo-ASR1 Is Predicted to Be Intrinsically Unstructured

Full-size tomato ASR1 can be expressed in Escherichiacoli as a water-soluble protein. Attempts to crystallizethe purified protein using a large number (.600) ofconditions were not successful. Primary amino acidsequence of ASR1 was thus analyzed using PONDR(Protein Disorder Predictor) VSL1 predictor (http://www.pondr.com) and the FoldIndex program (Priluskyet al., 2005; http://bioportal.weizmann.ac.il/fldbin/findex) based on the algorithm proposed by Uverskyet al. (2000a). Figure 3 shows that according to thesepredictions, ASR1 protein is mostly unfolded underphysiological conditions.

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Disordered proteins are relatively enriched in disorder-promoting amino acids (E, K, R, G, Q, S, P, and A) anddepleted in order-promoting residues (I, L, V, W, F, Y,and C; Romero et al., 2001). Four amino acid residuescontribute 61.7% of the total ASR1 protein residues.Three of these, Lys, Glu, and Ala (20, 18, and 15residues, respectively), are disorder promoting andthe fourth, His (18 residues), is neutral (other). Overall,67 out of the 115 amino acid residues of ASR1 aredisorder promoting. On the other hand, only 21 of the115 amino acid residues belong to the order-promotinggroup. ASR1 also lacks any W and C residues, definedas order-promoting residues. Figure 4 shows that thepercentage of disorder-promoting amino acid residuesin ASR1 is higher than average tomato encoded pro-teins (http://bioinformatics.weizmann.ac.il/blocks/help/CODEHOP/codons/tomato.codon.use). Likewise,order-promoting residues are more abundant in aver-age tomato proteins than in ASR1.

To experimentally test this prediction, full-lengthASR1 protein was expressed in E. coli and purifiedto homogeneity (Kalifa et al., 2004a). The Mr of theexpressed protein was confirmed by mass spectrom-etry (Rom et al., 2006). The purified 13.1-kD tomatoASR1 protein migrates in SDS-PAGE as an 18-kDprotein (Kalifa et al., 2004a; Rom et al., 2006) due toits high content of charged residues. Migration anom-aly in SDS-PAGE was demonstrated for other highlycharged proteins, many of which are natively un-folded (e.g. Weinreb et al., 1996).

Gel Filtration Analysis

The Stokes radius (RST) of purified ASR1 was deter-mined using gel filtration chromatography usingHiLoad 16/60 Superdex 200 column. The columnwas calibrated with Blue dextran (eluted at exclusionvolume), bovine serum albumin (RST 5 35.5 A), oval-bumin (RST 5 30.5 A), lysozyme (RST 5 19.8 A), andAMP (eluted at total volume). The column was equil-

ibrated and eluted with 50 mM Tris-HCl, pH 7.5, 100mM NaCl buffer at a flow rate of 0.5 mL/min. Elutiontimes for the calibration standards were 64, 106, 116,142, and 167 min, respectively. ASR1 protein eluted at124 min. Including zinc in the elution buffer resultedin protein precipitation within the column. Using theelution volumes of ASR1 and standards, the RST of 27.4A was determined for ASR1 (Table I). Uversky (2002)developed a set of equations for calculating the pre-dicted RST of polypeptides of a given Mr, assumingdifferent conformations of globular proteins. Usingthese equations for calculating the RST of a 13,121-Dpolypeptide, our data suggests that without zinc, theASR1 protein cannot be a globular protein but ratherexists in an unfolded protein (Table I).

Far-Ultraviolet Circular Dichroism Analysis

Circular dichroism (CD) is often used for the assess-ment of the fraction of structural components within a

Figure 1. Sedimentation equilibrium analyses of ASR1. Full-length ASR1 (0.2 mM) in buffer containing 20 NaPi pH 7.2 and 0.1 M

NaCl buffer (A) or in buffer containing 1 mM ZnCl2 (B) was brought to equilibrium at 4.0�C at three rotors speeds: A, 10,000 (red),14,000 (green), and 18,000 rpm (blue); B, 10,000 (red), 12,000 (green), and 14,000 (blue). Protein concentrations weremeasured by UV absorbance. Best fits in terms of two ideal solutes are shown as solid black lines through the correspondingsymbols.

Figure 2. Chemical cross-linking of ASR1. Purified full-size (WT ASR1)and truncated (ASR1D61-115) proteins were incubated in a mixwithout (2) or with (1) added 0.5 mM EGS. Mixes were treated withSDS sample buffer, resolved by SDS-PAGE, and stained with CoomassieBlue.

Structural Investigation of the Stress Protein ASR1

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given protein. The far-UV CD spectrum of purifiedASR1 (Supplemental Fig. S1) has a maximum negativeellipticity at 205 nm and low ellipticity around 215 to225 nm, which indicates a heavily disordered proteinstructure (Zeev-Ben-Mordehai et al., 2003). Further-more, no significant ellipticity was observed in thenear UV, suggesting that unlike typical globular pro-teins, in ASR1 there is no hydrophobic core containingoriented aromatic residues.

Dynamic Light Scattering Studies

Dynamic light scattering (DLS) is used to determinethe hydrodynamic radius of macromolecules. A hy-drodynamic radius of 3.01 nm was determined forASR1 without added zinc (Table II). This radius cor-responds to a 44.3-kD globular protein (3.4 times themass of monomeric ASR1) and is consistent with anunfolded 13.1-kD protein (Table I). Addition of 1 mM

ZnCl2 resulted in an increase of the hydrodynamicradius of the protein to a value corresponding toglobular protein of 100.2 kD. About 1.2% of the proteinanalyzed in the presence of zinc was aggregated witha calculated mass of about 9,000 kD. Similar hydrody-namic values were determined at pH 7 (data notshown). These results suggest that monomeric anddimeric ASR1 are not compact structures, because thehydrodynamic values determined for both forms arelarger than those expected for a tightly packed glob-ular protein.

Protein Structure Analysis by FTIR Spectrometry

Secondary protein structure can be estimated bymonitoring the FTIR amide I band (1,600–1,700 cm21).As distinct protein structural elements have specificpeaks, the fraction of major structure elements in theprotein can be determined by peak analysis of theobtained FTIR spectrum (Byler and Susi, 1986; Pribicet al., 1993). Freeze-dried ASR1 was dissolved in D2Oto avoid interference of the HOH vibration with amid Iband (Sieler and Schweitzer-Stenner, 1997). In theabsence of zinc, the FTIR spectrum displayed a peak

Figure 3. Prediction of the folding state of ASR1. A, ASR1 orderprediction via the VSL1 program of PONDR using the default param-eters. Values smaller and larger than 0.5 represent ordered andnonordered protein, respectively. B, ASR1 folding prediction via theFoldIndex program using the values predicted window 5 10 and step 5 1.Positive and negative numbers represent ordered and nonordered pro-tein, respectively. C, The primary amino acid sequence of ASR1. Aminoacids suggested in B as being ordered and nonordered regions areshown in regular and bold characters, respectively.

Figure 4. Percentage of amino acid residues in ASR1 and generaltomato proteins. The content of amino acid residues in tomato ASR1(gray bar) and in general tomato proteins (black bar) is plotted. A,Individual amino acid residues. B, Grouped residues according to theclassification as order or disorder promoting (Romero et al., 2001).

Goldgur et al.




at 1,642 cm21 (Fig. 5A), which corresponds to thesignature of a random coil (Byler and Susi, 1986;Krimm and Bandekar, 1986; Surewicz and Mantsch,1988; Bandekar, 1992; Surewicz et al., 1993). Dataanalysis suggests that the peak assigned for a disor-dered structure represents over 50% of the protein(Fig. 5A; Table III). Moreover, about 27% of the proteinis assigned to be in turn structure (1,680 and 1,693cm21 peaks). The 1,664 cm21 peak (13.2% of the totalarea) can be assigned to either turns (Byler and Susi,1986) or to a 310 helix (Surewicz et al., 1993). Nosignificant peak for b-strands was observed. Additionof zinc resulted in FTIR spectral changes for ASR1. Themajor (56.6%) 1,642 cm21 and minor (13.2%) 1,664cm21 peaks corresponding to a disordered or a turn/310 helix structure, respectively, are diminished andreplaced by peaks at 1,657 cm21 (39.9%) and 1,626cm21 (16.6%) in the presence of zinc, corresponding,respectively, to a-helix and b-strands structures (Fig.5B; Table III). The relative intensities of the other fittedpeaks revealed minor changes. Drying the ASR1 prep-aration resulted in similar changes of the observedspectrum to that observed for soluble ASR1 in thepresence of added zinc (Fig. 5C). Drying ASR1 from azinc-containing medium did not result in furtherchanges (Fig. 5D).

Thermal Stability of ASR1

Differential microcalorimetry (DSC) was used tomonitor temperature-induced changes in the structureof ASR1 plus and minus added zinc. No significantphase changes were observed in ASR1 solution with-out added zinc, over a temperature range of 4�C to90�C (Fig. 6). On the other hand, zinc binding to ASR1displayed structural heat denaturation at 76�C (Fig. 6),indicative of an ordered structure below this temper-ature.

Cytosolic ASR1 Is in Unstructured State

Unfolded proteins have increased sensitivity to pro-teases, and we showed that the susceptibility of puri-

fied recombinant ASR1 to a number of proteases isdecreased in the presence of zinc (Rom et al., 2006).Here, we analyzed the proteolytic susceptibility ofcytosolic tomato ASR1 without or with added zinc.Leaf soluble ASR1 is rapidly degraded by endogenoustomato proteases and by added trypsin (Fig. 7A).Addition of zinc to the tomato leaf soluble proteinfraction stabilized ASR1 and decreased markedly itsproteolytic degradation. This is consistent with foldingof the ASR1 protein. The protease effect is specific toASR1, as the proteolytic activities had only a marginaleffect on the overall electrophoretic protein pattern inthe extract (Fig. 7B). Moreover, the concentrations ofzinc used in this experiment did not inhibit the activityof trypsin (Rom et al., 2006).

ASR1 Is Present in Tomato Seeds and Pollen Grains

To address the possible physiological relevance ofdesiccation on inducing folding of ASR1 (Fig. 6C), weused anti-ASR1 antiserum to probe protein extractsprepared from tomato pollen grains and from fullydeveloped tomato seeds. Seeds were isolated fromfully ripened tomato fruit and rinsed thoroughly toremove residual loculus tissue, and pollen grains werecollected from tomato flowers. Figure 8 shows thatfully developed pollen grains and tomato seeds thatcontain very little water have high levels of ASR1protein (comparable to that in salt-stressed leaf tissueon the basis of fresh weight). Oligomeric form of ASR1is also observed in the leaf extract.

DISCUSSION

ASR1 as Hydrophilin

Hydrophilins are proteins defined by high hydro-philicity index and Gly content (.1.0% and .6%,respectively; Garay-Arroyo et al., 2000). Hydrophilinsare found mainly in plants, bacteria, and yeast (Sac-charomyces cerevisiae). They represent a small fraction(,0.2%) of the genome and are suggested to be apredictor for responsiveness to hyperosmosis (Garay-Arroyo et al., 2000). ASR1 meets with the hydrophilincriteria in that 7% of its amino acid residues are Gly,and the average protein hydrophilicity index is 1.17.Late embryogenesis abundant (LEA) proteins com-prise the largest group of hydrophilins. Although

Table I. Observed and calculated RST for ASR1

The RST of ASR1 was determined by gel filtration as described in‘‘Materials and Methods.’’

Analysis Sample/Form RST

A

Experimental 27.4Calculateda Native globular 18.5

Native molten globule 21.0Native premolten globule 25.4Denaturated unfolded (urea) 31.4Natively unfolded premolten globule 30.1Natively unfolded random coil 26.3

aRST of a 13,121-D protein in various conformational states wascalculated using equations developed by Uversky (2002).

Table II. DLS

ASR1 was solubilized in buffer containing 50 mM Tris-HCl, pH 7.5,and 100 mM NaCl. ZnCl2 was added to a final concentration of 1 mM.Samples were prepared and analyzed as described in ‘‘Materials andMethods.’’

[ZnCl2] Hydrodynamic Radius Total Mass

mM nm %

0 3.01 1001 4.26 98.8





ASR1 is not a LEA protein, because it lacks LEAsignatures (Wise and Tunnacliffe, 2004), it shares sim-ilarities with some LEA proteins; for example, the ex-pression of ASR1 and LEA is increased under osmoticstress. Moreover, some LEA proteins (e.g. proteinsfrom LEA subgroups 1a, 2a, 3b, and 6) are DNA-binding proteins (for review, see Wise and Tunnacliffe,2004).

ASR1 as Intrinsically Unstructured Protein

The ASR1 polypeptide chain is predicted to be in-trinsically unstructured (Figs. 3 and 4). It is relativelypoor in order-promoting hydrophobic amino acidresidues and enriched in disorder-promoting chargedamino acid residues. Uversky et al. (2000a) did a com-parative study of known natively unfolded proteins.They showed that most natively unfolded proteinshave low Mrs with less than 150 amino acid residues.ASR1, a 115-residue protein, conforms to this sizegroup. However, most natively unfolded proteinshave either an acidic or basic isoelectric pH, whereasthe calculated pI for ASR1 is almost neutral. Accordingto Uversky et al. (2000a), 130 different nonhomologousproteins in the Swiss Protein database were predicted

to be natively unfolded on the basis of length, lowmean hydrophobicity, and high net charge. TomatoASR1 was one of their predicted proteins.

Our data confirmed that Apo-ASR1 protein is non-structured in solution, as demonstrated by gel filtra-tion (Table I), DLS (Table II), CD (Supplemental Fig.S1), FTIR (Fig. 5A), and microcalorimetry (Fig. 6).Thus, in the absence of added zinc, ASR1 may beadded to the growing list of unstructured or poorlystructured small proteins that possess biological rolesrelated to water stress. For example, certain (thoughnot all) dehydrins were shown to be disordered undernative conditions (Eom et al., 1996; Lisse et al., 1996;Soulages et al., 2003; Mouillon et al., 2006). Near-infrared spectroscopy analysis of a number of proteinsof similar Mr range with ASR1 suggested that freeze-dried proteins mostly maintain spectral characteristicsof native structure (Izutsu et al., 2006). Thus, the freezedrying step in the preparation of ASR1 is not likely toinduce any major structural changes. Possible minorchanges resulting from freeze drying are expected tobe reversible upon redissolving the dried protein. DSCscans of group 1 LEA protein from pea (Russouw et al.,1997) and of group 1 (Soulages et al., 2002) and group 2(Soulages et al., 2003) LEA proteins from soybean

Figure 5. FTIR spectroscopy of ASR1.Freeze-dried ASR1 was solubilized inD2O supplemented with zero (A and C)or 1 mM (B and D) ZnCl2. FTIR spectra ofASR1 in solution soluble (A and B) or inair-dried preparation (C and D) weredetermined. Second derivatives of spec-tra (top) were used to obtain the differentband positions and in peak fitting.

Goldgur et al.




(Glycine max) showed no detectable high temperaturepeak, suggesting that these proteins failed to undergodetectable heat denaturation. EBM-1, a group 1 LEAprotein from carrot (Daucus carota), was suggested tobe unstructured in solution (Eom et al., 1996). Thenative form of DSP16, a dehydrin-like protein fromCraterostigma plantagineum, displayed no definedthree-dimensional structure (Lisse et al., 1996). Thenematode LEA protein homolog AavLEA1 was shownto be unfolded in solution (Goyal et al., 2003), and theD-7LEA protein from purified Typha latifolia pollenwas shown to be highly unfolded in solution (Wolkerset al., 2001). Finally, a large fraction of the soluble ASR1from tomato leaf exhibits an unfolded secondarystructure as shown by increased protease sensitivitythat could be reduced upon addition of zinc to the leafhomogenate (Rom et al., 2006).

Zinc Binding and Desiccation Induce Order andDimerization in ASR1 Protein

FTIR spectral analysis suggests that in the presenceof Zn21, ASR1 gains more a-helix and b-strand do-mains (Fig. 5; Table III), which implies a more highlyordered polypeptide structure. Zinc-dependent or-dered protein structure was also supported by micro-calorimetry (Fig. 6), where the peak of heat absorbancewas only observed in the presence of zinc. Neverthe-less, the ordered ASR1 dimers have a rather non-compact structure as determined by DLS (Table II).The free concentration of Zn21 in the reaction mixes ismuch smaller than that of the added ZnCl2 probablydue to sequestering of zinc ions by the buffer used(Dawson et al., 1986). The ability of bound zinc toaffect more structural order to the ASR1 polypeptidemonomer, apart from promoting homodimerization, issupported by the recent findings of Rom et al. (2006),where the presence of zinc reduced the sensitivity ofASR1 to protease digestion. Rom et al. (2006) alsoreported other zinc-binding phenomena relevance toASR1-DNA interactions. The importance of zinc inmediating protein structure pertinent to its functionhas been further strengthened by studies on humanprothymosin a, a protein characterized as natively un-

folded (Uversky et al., 2000b) and also by zinc-drivenfolding and oligomerization of g-carbonic anhydrase(Simler et al., 2004). Folding of apo-metalloprotein hasbeen reported to be induced by the binding of bivalentions, including zinc (Ejnik et al., 2002), and the foldingand stability of the nuclear hormone receptor DNA-binding domain was shown to be zinc dependent(Low et al., 2002). Moreover, the folding of a singlezinc-finger domain was observed to be dependenton binding of zinc ion (Frankel et al., 1987; Parragaet al., 1988). The concentration of free zinc in the cellsof biological organisms is not known (Outten andO’Halloran, 2001; Rutherford and Bird, 2004) and isestimated to be in the picomolar range. This is lowerthan the dissociation constant of zinc binding to ASR1(Kalifa et al., 2004a; Rom et al., 2006). Thus, the extentof zinc binding is expected to be dependent on the freezinc concentration.

We found that desiccation of an ASR1 solution alsoinduced increase of structure of the protein (Fig. 5;

Figure 6. DSC analysis of ASR1. Heat absorbance of a solutioncontaining purified ASR1 (0.2 mg/mL) in 20 mM HEPES-NaOH, pH7.5 buffer without (dashed line) or with 0.5 mM (solid line) ZnCl2 wasanalyzed using MicroCal VP-DSC micro calorimeter.

Table III. FTIR amide I components and their relative areas of ASR1 samples

Relative areas of the peak fitting analysis shown in Figure 5.

Wavenumber Peak

Area

ASR1 in Solution Dried ASR1

2Zn21 1Zn21 2Zn21 1Zn21

cm21 % of Total

1,610 0 4.8 2.8 3.81,626 0 16.6 20.3 19.11,642 56.6 10.0 11.7 15.91,657 0 39.9 41.5 39.41,664 13.2 0 0 01,680 18.9 17.3 15.6 12.11,693 8.5 7.3 5.5 7.61,710 2.8 4.0 2.5 2.0





Table III). Adding zinc had no further effect on thelevel of the obtained degree of order. In similar obser-vations, the D-7LEA protein purified from T. latifoliapollen was shown to be highly unfolded in solution(Wolkers et al., 2001), but upon drying, the proteinconformation exhibited a largely a-helix structure. More-over, the nematode LEA protein homolog AavLEA1was shown to be an unfolded protein in solution(Goyal et al., 2003), whereas desiccation of the proteininduced folding and increased structure. We detectedhigh levels of ASR1 in fully developed tomato seedsand in pollen grains (Fig. 8). The lily ASR1 orthologLLA23 was isolated from desiccating pollen grains(Wang et al., 1996, 1998). The developmental processesof both seed and pollen maturation involve desicca-tion. These results suggest that order induced bydesiccation might be a general mechanism of actionof dehydrin proteins that are involved in water-stressresponse and tolerance.

Analytical ultracentrifugation showed that in theabsence of zinc, ASR1 is a monomer (Fig. 1A). How-ever, in the presence of zinc ions, the protein becomes

homodimeric (Fig. 1B), suggesting that the increasedorder in the primary structure of the protein observedby FTIR (Fig. 5) promotes the dimerization of ASR1.ASR1 dimerization was also shown by chemical cross-linking (Fig. 2). Even the purified amino terminaldomain (residues 1–60) of ASR1 formed dimers (Fig.2). This truncated ASR1 portion bound two zinc ions(Rom et al., 2006). The zinc-dependent dimerization isalso supported by the increase of the hydrodynamicradius measured using DLS (Table II). The dimeriza-tion was also observed by chemical cross-linking evenin the absence of added zinc (Figs. 2). Although ana-lytical ultracentrifugation experiments were carriedout using higher protein concentrations than thoseused in the chemical cross-linking experiment, resid-ual zinc contaminations might be the basis of dimer-ization under low protein concentrations. Indeed, lowamounts of tightly bound zinc were detected in ASR1preparations even in the absence of added zinc (Romet al., 2006). Furthermore, it is known that chemical cross-linkers can be used to trap transient association betweenpolypeptides (Melcher and Xu, 2001), suggesting thatzinc stabilizes the interactions between the ASR1 mono-mers, which might associate only weakly in its absence.Zinc-dependent oligomerization of the amino terminalportion of ASR1 protein was also observed by analyticalultracentrifugation (data not shown), suggesting that thetightly bound pair of zinc ions (Rom et al., 2006) encour-ages homodimer formation.

Dimerization of protein disulfide isomerase is azinc-dependent process (Solovyov and Gilbert, 2004).Furthermore, cadmium ions were shown to induce thefolding and dimerization of a designed metalloprotein(Kharenko and Ogawa, 2004), and trimerization of anAla-containing peptide was shown to be mediated byzinc ions (Liu et al., 2003). A unique zinc-binding sitewas demonstrated in the x-ray structure of homotri-meric Apo2L/TRAIL protein (Hymowitz et al., 2000).This homotrimer contains a single zinc ion buried atthe trimer interface in a charge-shared coordinationbetween the three monomers.

Figure 7. Protease sensitivity of soluble tomato leaf ASR1. The 0.1-mLsamples of leaf total soluble protein fraction were incubated for 30 minat the indicated temperature, without or with 0.6 mM ZnCl2 and theindicated amounts of trypsin. SDS-PAGE sample buffer was added to afinal 23 concentration, proteins were heat denaturated, resolved onSDS-PAGE, and electroblotted onto nitrocellulose. The membrane wasstained with Ponceau S and probed with anti-ASR1. A, Western-blotanalysis using anti-ASR1. B, Ponceau S stain of the nitrocellulosemembrane. C, Quantification of the signals shown in A.

Figure 8. Western-blot analysis of tomato seed and pollen proteins.Seed and leaf acid soluble protein extract and pollen total proteinextract were resolved by SDS-PAGE, blotted onto nitrocellulose mem-brane, and probed with anti-ASR1. The 10-mL extracts were loaded oneach lane (equivalent for protein extracted from 25 mg of each seed andleaf tissue and 2 mg of pollen grain). L, Leaf; S, seed; P, pollen.

Goldgur et al.




With regards to the observation of ASR1’s zinc-dependent DNA-binding capability (Kalifa et al.,2004a), a dimeric protein structure is a commonlyobserved motif with DNA-binding proteins in general(see Burley and Kamada, 2002). For example, thefolding of intrinsically disordered C terminus of anucleoprotein from measles virus depends on thisprotein’s binding to a second protein (Bourhis et al.,2004). In Kalifa et al. (2004a), certain oligonucleotidesselected as binding partners for ASR1 in vitro con-tained two copies of the ASR1 consensus DNA-bindingdomain, supporting our experimental observation thatactive ASR1 is homodimeric.

CONCLUSION

Apo-ASR1 protein is intrinsically disordered (Fig.9A). ASR1 gain order upon binding of zinc, most likelyto the two zinc-binding sites in the N-terminal domain(Fig. 9B; Rom et al., 2006) or possibly upon desiccation.Zinc is also involved in the DNA-binding activity ofASR1, possibly at another binding site(s) localized atthe central part of the polypeptide (Fig. 9C; Rom et al.,2006). However, these ASR1 structural results alsoimply that prediction methods for disordered struc-tures of proteins are too simplistic, because they do nottake account of ligand binding to other domains orprotein-protein interactions. The ASR1 protein wasshown to be localized both in the cytosol and nucleusof tomato leaf cells (Kalifa et al., 2004a). We proposethat the nuclear-located DNA-bound ASR1 is ordered,whereas the cytosol pool of the protein might be ateither structure. The secondary and tertiary structuresof ASR1 protein as a function of its location in eachsubcellular compartment have yet to be determined.Soluble tomato ASR1 is mainly unordered, because itis highly susceptible to protease degradation (Fig. 7).Upon zinc binding, the soluble ASR1 becomes moreresistant to protease, indicating that a structuralchange has occurred. Zinc binding was shown todecrease the protease sensitivity of purified recombi-nant ASR1 (Rom et al., 2006). On the other hand,nuclear-located DNA-bound ASR1 is ordered, becauseDNA binding is dependent on zinc (Kalifa et al.,2004a). Alternating between unfolded and orderedstructures may comprise a means of regulating activityof the ASR1 protein through the linkage betweendesiccation and zinc binding. We propose that the

DNA-bound ASR1 is folded, because DNA binding iszinc dependent, whereas the cytosol pool comprisedof unfolded protein (Fig. 7). We cannot rule out that afraction of the cytosolic ASR1 protein is ordered.

MATERIALS AND METHODS

Plant Materials

Tomato (Solanum lycopersicum) seedlings were grown in vermiculite, as

previously described (Kalifa et al., 2004b). One-month-old seedlings were

watered with fertilizer solution without or with added 0.15 M NaCl, at a

volume 5 times larger than pot volume. Leaves were collected 2 d later. Fully

developed seeds were collected from well-ripened tomato fruit, washed

thoroughly to remove loculus tissue, and blotted dry on filter paper. Acid

soluble proteins were extracted, as previously described (Kalifa et al., 2004b).

Pollen grains were collected from greenhouse-grown tomato plants. A 23

SDS-PAGE sample buffer (Laemmli, 1970) was added (5 mL/g pollen).

Samples were heated for 30 min at 70�C and centrifuged for 15 min at

12,000g at room temperature.

Protease Sensitivity Assay

Leaves of salt-treated tomato plants were homogenized in ice-cold 50 mM

HEPES-NaOH, pH 7.5 buffer, using 5 10-s bursts of KINEMATICA POLYTRON

(Brinkmann Instruments) homogenizer. The homogenate was filtered thor-

ough three layers of Miracloth (Calbiochem) and centrifuged for 15 min at

12,000g at 4�C. The supernatant was divided into two portions. ZnCl2 was

added to one portion to a final volume of 0.6 mM. Then 0.1 aliquots were

incubated at 37�C for 30 min in the presence of the indicated amounts of

trypsin. Seventy microliters 53 SDS-PAGE sample buffer was added, and

mixes were heated at 70�C for 30 min and loaded immediately onto polyac-

rylamide gels.

ASR1 Expression and Purification

Full-length nontagged ASR1 protein was expressed in Escherichia coli and

purified to homogeneity by metal chelating chromatography, as described

(Kalifa et al., 2004a; Rom et al., 2006).

Protein Electrophoresis and Western-Blot Analysis

Denaturating PAGE (15% [w/v] SDS-PAGE) was run in high concentra-

tions of Tris for improved resolution of polypeptides with low Mr (Fling and

Gregerson, 1986). Gels were stained with Coomassie Blue or electroblotted

onto nitrocellulose membranes. ASR1 protein was detected using the previ-

ously described anti-ASR1 antiserum (Amitai-Zeigerson et al., 1995).

Gel Filtration Chromatography

Purified ASR1 and the protein standard mixture were loaded onto a

HiLoad 16/60 Superdex 200 column (Pharmacia). The column was preequili-

brated and eluted with buffer containing 50 mM Tris-HCl, pH 7.0, and 100 mM

NaCl at a flow rate of 0.5 mL/min. The elution volume (Ve) was monitored by

A280. The Ve for a particular molecular species was then converted to Kav by the

following equation:

Kav 5 ðVe 2 VoÞ=ðVt 2 VoÞ;

where Vo and Vt are exclusion and total volumes, taken as the elution volumes

of dextran blue and AMP, respectively. RST was estimated using a linear

calibration plot of RST versus (2log Kav)1/2 (Siegel and Monty, 1966). The

following standard proteins were used: bovine serum albumin (RST 5 35.5 A),

ovalbumin (RST 5 30.5 A), and lysozyme (RST 5 19.8 A).

DLS

Lyophilized samples of ASR1 were dissolved in 20 mM Tris-HCl, pH 7.5,

100 mM NaCl buffer to a final concentration of 1 mg/mL. A 100-mM ZnCl2 solu-

tion was added to aliquots of both solutions to the final concentration of 1 mM.

Figure 9. Scheme describing changes in ASR1 protein order andoligomeric state induced by binding of zinc ions and DNA.





All samples were filtered through 0.2-mm filters and centrifuged at 16,000g for

10 min immediately before measurements. The measurements were carried out

using DynaPro-801 DLS instrument (Protein Solutions). The wavelength of the

incident light was 780 nm. The autocorrelation function of the scattered light

intensity is used to calculate the diffusion coefficient DT that is converted to the

hydrodynamic radius using the Stokes-Einstein equation:

RH 5 kBT=6phDT;

where kB is the Boltzman’s constant, T is the abolute temperature, and h is the

solvent viscosity.

CD

Purified ASR1 protein was dialyzed against 20 mM HEPES-NaOH, pH 7.5.

Mixtures contained ASR1 protein in 20 mM HEPES-NaOH, 20 mM NaCl, and

the indicated concentrations of ZnCl2. pH values of the resulting solutions

were readjusted to 7.5. CD spectra were recorded at room temperature using a

Jasco Circular Dichroism Spectroscope (model J-715).

DSC

DSC studies were performed using a MicroCal VP-DSC micro calorimeter.

Heat absorbance by purified ASR1 (0.2 mg/mL) in 20 mM HEPES-NaOH, pH

7.5, in the presence of the indicated concentrations of ZnCl2 was scanned from

4�C to 90�C at a scanning rate of 0.5�C/min. The pH of samples containing

zinc was corrected to 7.5.

FTIR Spectroscopy

Purified ASR1 was dialyzed against 20 mM HEPES-NaOH at pH 7.0, 7.5, or

8.0, freeze dried, and redissolved in D2O at one-third of the original volume.

ZnCl2 was added to specific samples. Samples were placed on the surface of

ZnSe crystal, and spectra were recorded using a Bruker Equinox 55 spec-

trometer (Bruker Optics). Protein drying experiments were done by air drying

the D2O-dissolved sample on spotted on the surface of ZnSe crystal at room

temperature. The spectra shown are average values of triplicate runs, each

composed of 120 measurements. Parallel preparations showed that addition

of zinc to the protein samples reduced the pH by less than 0.5 pH units.

Second derivative analyses of spectra and peak fitting were performed using

PeakFit software (Systat Software).

Analytical Ultracentrifugation

ASR1 (0.2 mM) was dialyzed extensively against a buffer of 20 mM NaPi, pH

7.2, 0.1 M NaCl. The sample was divided in half and ZnCl2 (0.1 M) was added

to one portion to a final concentration of 1 mM. Sedimentation equilibrium

analysis was conducted at 4�C with a Beckman Optima XL-A analytical

ultracentrifuge. Samples (160 mL) were studied at different rotor speeds:

10,000 to 18,000 rpm. Data were acquired as an average of eight absorbance

measurements at 280 nm and a radial spacing of 0.001 cm. Equilibrium was

achieved within 24 h. Due to the presence of a small amount of aggregated

protein, data collected at three different rotor speeds were analyzed simul-

taneously in terms of two noninteracting ideal solutes using SigmaPlot 8.0

(SPSS). Simultaneous, weighted, nonlinear least-squares fitting of the data sets

at each loading concentration was performed using a mathematical model of

the following form:

Ar 5 A0;1exp½HM1ð1 2 v1rÞðr22 r

2

oÞ�1 A0;2exp½HM2ð1 2 v2rÞðr22 r

2

oÞ�1 E;

where A0,1 and A0,2 are the absorbance of species 1and 2, respectively, at a

reference point ro, Ar is the absorbance at a given radial position r, H represents

v2/2RT, v is the angular speed in rads21, R is the gas constant, T is the absolute

temperature, and E a small baseline correction determined experimentally by

overspeeding. Residuals were calculated, and a random distribution of the

residuals around zero (60.02) was obtained as a function of the radius. Values

of the smaller mass, M1, were obtained from the buoyant molecular mass,

given as M1(1 2 v1r), and calculated using densities, r, at 4�C obtained from

standard tables. A value of v1 of 0.7360 mL g21 was calculated for ASR1 based

on the amino acid composition using consensus data for the partial specific

molar volumes of amino acids (Perkins, 1986). Molecular masses, M2, of the

larger species or aggregate were calculated in a similar fashion.

Sequence data from this article can be found in the GenBank/EMBL data

libraries under accession number L08255.

Supplemental Data

The following materials are available in the online version of this article.

Supplemental Figure S1. CD spectrum of ASR1.

ACKNOWLEDGMENTS

The authors thank Dr. Sofiya Kolusheva for helping in the microcalorim-

etry studies and Dr. Peter S. Coleman for critical reading of the manuscript.

Received November 14, 2006; accepted December 3, 2006; published

December 22, 2006.

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Desiccation and Zinc Binding Induce Transition of Tomato ... · drought and salinity (Yang et al., 2005). Maize (Zea mays ) ASR1 was proposed as a candidate gene for quantitative